WO2022179648A1 - Polypeptides imitant les épitopes de glycoprotéines env du vih-1 à boucle v3 et mper - Google Patents

Polypeptides imitant les épitopes de glycoprotéines env du vih-1 à boucle v3 et mper Download PDF

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WO2022179648A1
WO2022179648A1 PCT/CZ2022/050017 CZ2022050017W WO2022179648A1 WO 2022179648 A1 WO2022179648 A1 WO 2022179648A1 CZ 2022050017 W CZ2022050017 W CZ 2022050017W WO 2022179648 A1 WO2022179648 A1 WO 2022179648A1
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seq
hiv
polypeptide according
sequence
virus
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PCT/CZ2022/050017
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Petr Maly
Milan RASKA
Milan Kuchar
Petr Kosztyu
Jiri Cerny
Veronika Daniel Liskova
Hana PETROKOVA
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Univerzita Palackeho V Olomouci
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Priority to CN202280017607.3A priority Critical patent/CN116964080A/zh
Priority to US18/278,208 priority patent/US20240226269A9/en
Priority to BR112023016617A priority patent/BR112023016617A2/pt
Priority to EP22712245.4A priority patent/EP4298119A1/fr
Publication of WO2022179648A1 publication Critical patent/WO2022179648A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4716—Muscle proteins, e.g. myosin, actin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to polypeptides derived from the structure of human contractile protein myomesin-1 domain.
  • the polypeptides of the present invention include three groups of polypeptides designated MLA, MLB and MLD targeted to 10E8, PGT126, and PGT121 broadly neutralizing antibodies (bNAbs) against HIV-1 virus, respectively.
  • the polypeptides recognize a paratope of the particular HIV-1 bNAb and mimic the cognate epitopes of Env glycoprotein and are, therefore, suitable as immunogens for stimulation of production of HIV- 1 -neutralizing antibodies and for the development of a vaccine preventing HIV infection.
  • bNAbs broadly neutralizing antibodies
  • Identification and characterization of highly potent bNAbs cloned from B-cells of elite neutralizers provide a molecular clue for designing new vaccine strategies.
  • the main target of neutralizing antibody on the HIV-1 virus surface is the envelope glycoprotein (Env), heterotrim er composed of three identical gpl20/gp41 subunits. Gpl20 protrudes from virus surface whereas gp41 is transmembrane subunit.
  • V2 bNAbs V2 loop of HIV-1 Env glycoproteins
  • V3 bNAbs V3 glycan
  • CD4 bNAbs CD4 binding site
  • MPER bNAbs membrane-proximal external region
  • Some of these bNAbs exhibit extraordinary breadth and potency and efficiently neutralize viral infection of host cells and represent exclusive candidates for vaccine design and therapy (Sok, D., and Burton, D.R. Recent progress in broadly neutralizing antibodies to HIV. Nat Immunol 19, 1179-1188, 2018).
  • the present invention provides polypeptides called herein ML A suitable for induction of HIV-1 virus neutralizing antibodies which targetoughsuper candidate“ bNAb 10E8 that is specific for MPER of gp41 subunit protruding from the viral envelope.
  • the present invention also provides polypeptides called herein MLB and MLD that mimic V3-loop epitopes of highly potent neutralizing antibodies PGT126 and PGT121, respectively.
  • the all polypeptides were obtained from a loop-randomized scaffold library designed on the structure of domain 10 of human contractile muscle protein myomesin-1.
  • the novel polypeptides are used as immunogens in experimental mice to stimulate the production of specific antibodies, the Env-specificity and virus -neutralizing activity of the hyperimmune sera tested on a panel of pseudotyped HIV-1 viruses of clades A, B, C, D and AE result in neutralization of most of the tested pseudoviruses in vitro.
  • the sequence of the parental human myomesin-1 domain 10 protein (PDB ID 6T30) is as follows: KSELAVEILEKGQVRFWMQAEKLSGNAKVNYIFNEKEIFEGPKYKMHIDRNTGIIEMFMEKLQD EDEGTYTFQLQDGKATNHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG (SEQ ID NO. 1)
  • amino acid residues in positions 21-24, 50-52 and 76-80 were randomized. These positions are shown in bold in SEQ IN NO. 1.
  • the present invention thus provides polypeptides having the length of up to 180 amino acids and containing a sequence selected from sequences identical or differing at most in 5 amino acids from the sequence:
  • X21X22X23X24 is selected from KAQQ (SEQ ID NO. 33), LSVF (SEQ ID NO. 34), ATPS (SEQ ID NO. 35), EIMW (SEQ ID NO. 36), DGSS (SEQ ID NO. 37), LLPL (SEQ ID NO. 38), WMWW (SEQ ID NO. 39), MNLY (SEQ ID NO. 40), MWRN (SEQ ID NO. 41), IMME (SEQ ID NO. 42), KHQL (SEQ ID NO. 43), HWQF (SEQ ID NO. 44), YAGN (SEQ ID NO. 45) and HGQW (SEQ ID NO. 46);
  • X50X51X52 is selected from RNT, IMF, GHE, PSW, RAN, YFW, ITL, QAM, DMR, WLW, QGE, VQY and VSL;
  • X76X77X78X79X80 is selected from SHHLG (SEQ ID NO. 47), FMLMM (SEQ ID NO. 48), VILIL (SEQ ID NO. 49), IVTPL (SEQ ID NO. 50), DFIIW (SEQ ID NO. 51), MWSE(deletion) (SEQ ID NO. 52), LYYAW (SEQ ID NO. 53), MMIEY (SEQ ID NO. 54), WMTQT (SEQ ID NO. 55), PQLWL (SEQ ID NO. 56), EPIFL (SEQ ID NO. 57) and QTATY (SEQ ID NO. 58), optionally further having an affinity tag attached to the N-terminus or to the C-terminus of the sequence.
  • X21X22X23X24 is selected from KAQQ (SEQ ID NO. 33), LSVF (SEQ ID NO. 34), ATPS (SEQ ID NO. 35), EIMW (SEQ ID NO. 36), DGSS (SEQ ID NO. 37), LLPL (SEQ ID NO. 38), WMWW (SEQ ID NO. 39), and MNLY (SEQ ID NO. 40);
  • X50X51X52 is selected from RNT, IMF, GHE, PSW, RAN, YFW, ITL and QAM;
  • X76X77X78X79X80 is selected from SHHLG (SEQ ID NO. 47), FMLMM (SEQ ID NO. 48), VILIL (SEQ ID NO. 49), IVTPL (SEQ ID NO. 50), DFIIW (SEQ ID NO. 51), MWSE(deletion) (SEQ ID NO. 52), LYYAW (SEQ ID NO. 53) and MMIEY (SEQ ID NO. 54).
  • MLA polypeptides having these variables are called MLA polypeptides.
  • X 21 X 22 X 23 X 24 is selected from MWRN (SEQ ID NO. 41), IMME (SEQ ID NO. 42) and KHQL (SEQ ID NO. 43);
  • X 50 X 51 X 52 is selected from RNT, DMR and WLW;
  • X 76 X 77 X 78 X 79 X 8 o is selected from IVTPL (SEQ ID NO. 50) and WMTQT (SEQ ID NO. 55).
  • X 21 X 22 X 23 X 24 is selected from HWQF (SEQ ID NO. 44), YAGN (SEQ ID NO. 45) and HGQW (SEQ ID NO. 46);
  • X 50 X 51 X 52 is selected from QGE, VQY and VSL;
  • X 76 X 77 X 78 X 79 X 80 is selected from PQLWL (SEQ ID NO. 56), EPIFL (SEQ ID NO. 57) and QTATY (SEQ ID NO. 58).
  • MLD polypeptides having these variables are called MLD polypeptides.
  • polypeptides of the present invention have the length of up to 180 amino acids and contain sequence selected from sequences identical or differing at most in 5 amino acids from the sequences:
  • KSELAVEILEKGQVRFWMQAKAQQGNAKVNYIFNEKEIFEGPKYKMHIDIMFGIIEMFMEKLQD EDEGTYTFQLQSHHLGNHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG SEQ ID NO. 3
  • KSELAVEILEKGQVRFWMQALSVFGNAKVNYIFNEKEIFEGPKYKMHIDRNTGIIEMFMEKLQD EDEGTYTFQLQFMLMMNHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG SEQ ID NO.
  • KSELAVEILEKGQVRFWMQAWMWWGNAKVNYIFNEKEIFEGPKYKMHIDITLGIIEMFMEKLQ DEDEGTYTFQLQLYYAWNHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG SEQ ID NO. 9
  • KSELAVEILEKGQVRFWMQAMNLYGNAKVNYIFNEKEIFEGPKYKMHIDQAMGIIEMFMEKLQ DEDEGTYTFQLQMMIEYNHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG SEQ ID NO.
  • sequences of the present invention are identical to sequence SEQ ID NO. 2 (with the listed variable regions), or to sequences SEQ ID NO. 3 to SEQ ID NO. 16, optionally further having an affinity tag attached to the N-terminus or to the C-terminus of the sequence.
  • sequences of the present invention differ in at most 5 amino acids from sequence SEQ ID NO. 2 (with the listed variable regions), or from sequences SEQ ID NO. 3 to SEQ ID NO. 16, and optionally further have an affinity tag attached to the N-terminus or to the C-terminus of the sequence.
  • the term “have a length of up to 180 amino acids” means that the sequence SEQ ID NO. 2 (with the listed variable regions), or sequences SEQ ID NO. 3 to SEQ ID NO. 16, may be extended on C-terminus or on N-terminus or on both termini with a maximum total of 69 amino acids (or 70 amino acids).
  • the length of the polypeptide may be up to 160 or up to 140 or up to 120 amino acids.
  • polypeptides of this invention preferentially bind to variable regions of HIV-1 broadly neutralizing monoclonal antibodies 10E8 (MLA polypeptides), PGT126 (MLB polypeptides) and PGT121 (MLD polypeptides) as demonstrated by a higher binding affinity to these bNAbs in comparison to IgG isotype control and by competition assays.
  • MAA polypeptides HIV-1 broadly neutralizing monoclonal antibodies 10E8
  • PGT126 MBB polypeptides
  • PGT121 MLD polypeptides
  • Use of polypeptides can be affected by a combination of an initial immunization dose and further booster doses, in which different versions of the polypeptides due to a substantial shape surface similarity can significantly increase production of neutralizing antibodies raised against the particular epitope- mimicking immunogen.
  • Affinity tags are typically used for purification of produced polypeptides, and then they can be cleaved or maintained in the sequence.
  • Polypeptide tags may include, for example: ALFA-tag, AviTag, C-tag, polyglutamate tag, polyarginine tag, E-tag, FLAG-tag, HA-tag, His-tag, Myc-tag, NE-tag, RholD4-tag, S-tag, Softagl, Softag3, Spot-tag, Strep-tag, T7-tag, TC tag, Ty tag, V5 tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Dog-Tag, SdyTag.
  • Preferred tags are poly(his), FLAG, AviTag, HA, Myc, S-tag or V5-tag.
  • Polypeptides in the present invention are appropriate for the use in pharmaceutical technology, especially as mimicking recombinant protein ligands exploitable for development of more efficient vaccine preventing HIV-1 virus infection.
  • helper proteins can be covalently linked to polypeptides forming a chimeric protein.
  • Further possibilities are to modify the mentioned polypeptides by an attachment of helper N- or C-terminal sequences (tags), which allow their specific detection or their oriented immobilization to surface of carries such as nanoliposomes with enhancement of immunization efficacy.
  • tags include, in preferred embodiments, affinity or detection tags such as poly(his), FLAG, AviTag, HA, Myc, S-tag or V5-tag.
  • Polypeptides in the present invention defined by amino acid sequence stimulate production of serum antibodies after being used for immunization of experimental animals. Hyperimmune sera of immunized animals suppressed infection of reporter cells by tested Env-pseudotyped viruses in the model system and this represents one of the key mechanisms of HIV-1 infection control and one of the aims for development of a preventative vaccine that is still not available in the market.
  • the major advantage of these polypeptides, in comparison to vaccines currently being tested is their mimicking complexity for three different HIV-1 Env epitopes located at MPER of gp 41 and V3-loop of gpl20. This allows neutralizing distinct prominent epitopes defined by the most efficient HIV-1 bNAbs 10E8, PGT121 and PGT126.
  • the present invention thus also provides a vaccine for prevention of HIV-1 virus infection, comprising polypeptide according to the present invention and/or conjugate according to the present invention as an active ingredient or as an auxiliary ingredient.
  • the vaccine may contain at least two polypeptides according to the present invention and/or at least two conjugates according to the present invention. Suitable auxiliary substances for formulation of vaccines are known in the art.
  • the present invention further provides a DNA sequence selected from the group comprising complementary DNA coding for the amino acid sequence of the polypeptides of the present invention
  • the present invention further includes the use of said DNA sequence for the preparation of polypeptides or recombinant proteins produced in bacterial, yeast, insect, mammal or human host cells, and also these host cells, containing at least one DNA sequence of the present invention.
  • FIG. 1 Production characterization of human myomesin-1 domain 10.
  • FIG. 1 Human Myomesin-1 Domain 10, a Loop-type Myomedin library concept.
  • the domain 10 is shown with the randomized residues indicated as black spheres.
  • B) A rotated view on domain 10 is shown in cartoon representation with the randomized residues indicated as black spheres.
  • FIG. 3 Binding of MLA protein variants to 10E8 bNAb, IgG isotype control and BSA.
  • Myomedin variants of eight selected MLA clones in the form of purified fusion proteins with N-terminal polyhistidine tag and C-terminal V5 tag produced in E.coli BL21 (DE3) were assayed in ELISA, the parental non-randomized Myomedin was used as a negative control.
  • FIG. 4 Immunization of experimental mice with a panel of MLA variants induced serum antibodies specifically recognizing HIV-1 Env on the virus surface.
  • Neutralization assays were performed using a set of HIV-1 Clade A, B, C, AE, and D pseudoviruses of Tier 2 or 3 with TZM-bl indicator cells. Serially diluted serum samples in duplicates were incubated with pseudoviruses. After the incubation pseudovirus with serum, TZM-bl cells at a density of 105 cells/ml were added, incubated, lysed, and after addition of substrate, luminescence was measured.
  • FIG. 6 An unrooted phylogenetic tree of 194 representative sequences from HIV-1 2019 Compendium of HIV-1 ENV genes (https://www.hiv.lanl.gov) enriched for 22 HIV-1 ENV sequences used in this analysis. HIV-1 strains are classified into four groups M, N, O and P. The major group M is further divided into nine genetically distinct subtypes A, B, C, D, AE, G, H, J, F and K. Each subtype contains hybrid viruses - circulating recombinant forms as a result of genetic material combination. For better orientation, only the 22 HIV-1 ENV sequences used in the analysis are visualized.
  • FIG. 8 Binding of MLD protein variants to PGT121 bNAb, IgG isotype control and BSA. Three selected MLD clones were assayed in ELISA. Each point is shown as the mean value of duplicate with standard deviation.
  • FIG. 9 Competition of MLB and MLD protein variants with HIV envelope glycoprotein gpl20 for binding to PGT126 or PGT121 bNAb in ELISA. Their binding to PGT121 or PGT126 bnAb, respectively, was visualized by the anti-Flag M2 Ab-HRP conjugate or in the case of biotinylated protein by streptavidin-HRP. Each point is shown as the mean value of triplicates with standard deviation.
  • FIG 10. Immunization of experimental mice with a panel of MLB variants induced serum antibodies specifically recognizing HIV-1 Env. Sera were collected and tested in their reactivity with multimerizing HIV-1 gpl20 clade B consensus coated on ELISA plates. Antibody titers of IgG isotype were measured in ELISA. Statistical comparison was performed by ANOVA Kruskal- Wallis test with Dunn's post-test (* P ⁇ 0.05).
  • Myomedins the polypeptides of the invention, derived from human Myomesin-1 domain 10, are called Myomedins.
  • Myomesin-1 protein (MYOM1) in E. coli with N-terminal 6xHis-tag and thrombin cleavage site.
  • the protein was purified by Ni-NTA agarose and size-exclusion chromatography and its X-ray structure was solved to 1.8 A resolution.
  • the identified structure was deposited to the Protein Data Bank under accession code (PDB ID) 6t3o.
  • Template DNA corresponding to the GenBank BC116183 was obtained from the Source BioScience (Nottingham, UK).
  • Myomesin domain 10 was amplified by pair of primers (forward MyomlO-F CATATGAAATCAGAGTTGGCAGTTGAAAT, SEQ ID NO.
  • the protein was purified using Ni-NTA agarose (Qiagen, Germany) under native conditions. Eluted protein fractions were pooled, concentrated and applied to the size-exclusion chromatography column Superdex 75 10/300 GL (GE Healthcare, UK) with running buffer 20mM Tris, 50mM NaCl pH 8.0. As documented in Fig. 1A, produced protein analyzed by SDS-PAGE gel electrophoresis corresponds to an expected molecular weight 16 kDa.
  • the crystal structure of the myomesin-1 domain 10 (PDB ID 6t3o) is formed by 111 amino acids, which constitute the 2-layer sandwich of the immunoglobulin-like domain with 7 antiparallel b-strands and a terminal a-helix (Fig. 2A, B).
  • the domain corresponds to the MY10 part of the myosin filament-linking protein myomesin-1 PDB ID 2y23 with 100% identity.
  • To select a set of myomesin residues suitable for randomization we have performed in silico mutability screening of all amino acid residues.
  • Myomedin combinatorial library was assembled by a series of three PCR using Phusion High-Fidelity DNA Polymerase (NEB, Massachusetts, USA) using a list of primers and adaptors (see Table 1. below). 1st PCR (annealing temperature 65°C, 10 cycles) with 100 mM oligonucleotides MYOM-LP nlF, MYOM-LP 2F and MYOM-LP_n2R resulted in 147 bp product, which was used in 2nd PCR (annealing temperature 59°C, 10 cycles) with 10 mM oligonucleotides MYOM-LP 1F and MYOM-LP 3R.
  • 3rd PCR with 10 pM oligonucleotides L-for and L-rev was used to complete Myomedin sequence (333 bp). Furthermore, two additional PCR was used, 4th PCR with primers JOIN-F (adding ribosome binding site RBS), and JOIN-R and finally 5th PCR with primers T7B (adding T7 promoter) and TolAk joining Myomedin and TolA spacer ( E . coli str. K-12). TolA spacer template for the 5th PCR was amplified using primers P7 link and TolA rev from isolated genomic DNA of E. coli. The resulted PCR product representing linear vector for in vitro transcription and translation (5'-T7 promotor-5 'stem loop-RBS- Myomedin-TolAk-3'stem loop-3') was extracted from agarose gel and purified.
  • Table 1 List of oligonucleotides for combinatorial library assembly and ribosome display. Trinucleotide mixture of 19 amino acid codons (Ella Biotech GmbH, Martinsried, Germany) was used for the synthesis of randomized positions (bold) of a coding DNA strand (X01) and the complementary DNA strand (R01).
  • Myomedin combinatorial library was used for in vitro transcription/translation and further ribosome display (RD) selection.
  • Three-round RD selections were performed, 96-well Polysorp plates (NUNC, Denmark) were coated by 10E8 IgGl diluted in coating 100 mM bicarbonate/carbonate solution (pH 9.6) at a concentration according to the adjusted stringency in each round of RD selection procedure: 1st round - 25 pg/mL, 2nd round - 10 pg/mL and 3rd round - 10 pg/mL.
  • Pre-selection procedure was performed in wells coated with human IgGl lambda antibody at a constant concentration of 25 pg/mL in each round.
  • Table 2 Table of MLA, MLB and MLD sequences. Sequence comparison of the protein variants with parental non-randomized MyoWT protein. Grey boxes indicate the loop stretches with 12 positions at which the residues of MyoWT were randomized. Other residues of the 111 amino acid Myomedin scaffold protein were not mutated in all shown binding proteins.
  • ELISA Cell lysates of E. coli clones producing Myomedin protein variants were prepared using sonicator (Misonix 3000).
  • Polysorp plate (NUNC) was coated with 10E8, PGT121 or PGT126 IgG (5 pg/mL) or IgGl lambda (5 pg/mL) in coating buffer (100 mM bicarbonate/carbonate solution, pH 9.6) at 7°C overnight. Next day, the plate was washed by PBST solution (PBS buffer containing 0.05% Tween, pH 7.4) and wells were blocked by PBSTB (PBS buffer pH 7.4, containing 0.05% Tween and 1% BSA).
  • PBST solution PBS buffer containing 0.05% Tween, pH 7.4
  • PBSTB PBS buffer pH 7.4, containing 0.05% Tween and 1% BSA
  • the lysate samples (diluted 33 x) purified protein variants as well as Myomedin-wt negative control diluted in PBSTB were applied. Binding of the Myomedin variants was detected using anti-V5 tag - HRP conjugate in PBSTB (1:10 000, Abeam, Cambridge, UK). Results were visualized by the enzymatic reaction of HRP with TMB-Complete 2 substrate (TestLine Clinical Diagnostics s.r.o., Brno, Czech Republic), the reaction was stopped by 2 M sulfuric acid and absorbance at 450 nm was measured. Myomedin variants induce serum antibodies recognizing Env glycoproteins on pseudotyped HIV-1 viruses.
  • MLA protein variants 016, 024, 025, 092, 093, 132, 158 and 159 were expressed and purified as recombinant proteins and used for immunization of experimental mice by i.d. route with Freund’s adjuvant by four consecutive antigen injections, according to the schedule presented in Figure 4A.
  • MPER-specific reaction was evaluated on pseudoviruses coated on ELISA panel as the presence of membrane surrounding the gp41 is necessary to MPER epitope.
  • Four pseudoviruses - one Clade C Env (ZM109F) Figure 4B
  • three Clade B Env AC10.0, QH0692, RHPA
  • the experiment identified significant binding for MLA 025, 092, 093, 158 and 159 at least with one pseudotyped virus (Figure 4B, C) when compared with wild-type Myomedin (MyoWT).
  • MyoWT did not elicit detectable Env-pseudovirus specific serum response.
  • ELISA competition assay where the binding of sera to selected pseudovirus from Clade B (here RHPA) competed with a serial dilution of 10E8 as an original specific target used for all MLA variants selection (Figure 4D). 10E8 in concentration 2pg/ml completely inhibited all tested hyperimmune sera reactivity.
  • mice Female BALB/c mice, 6-8 weeks old, 18-22 g (AnLab, Brno, Czech Republic), were used for all immunization experiments. Animals were housed under standard conditions according to ARRIVE guidelines. The immunization experiments were approved by the Ethics Committee of the Faculty of Medicine and Dentistry (Palacky University, Olomouc, Czech Republic) and the Ministry of Education, Teen and Sports, Czech Republic (MSMT -9487/2019-3). All mice were immunized four times. Pre- immune (naive) sera were collected before the first immunization.
  • mice were immunized by the administration of four doses of individual MLA variants including wild- type (MyoWT). Each group consisted of five animals.
  • Neutralization assays were performed using a set of HIV-1 Clade A, B, C, AE, and D pseudoviruses of Tier 2 or 3 with TZM-bl indicator cells. Serially diluted serum samples in duplicates were incubated with pseudoviruses. The pseudoviruses load was set to achieve approximately 150 000 RLU in 150 m ⁇ of DMEM in the absence of sera. After the incubation pseudovirus with serum, TZM-bl cells at a density of 105 cells/ml were added, incubated, lysed, and after addition of substrate, luminescence was measured. Results are indicated in Fig.5. The 22 HIV-1 ENV sequences used in the analysis are visualized in Fig. 6.
  • Reactivity of binding antibodies targeting Env was measured by ELISA using pseudotyped viruses prepared for virus neutralization assay (MLA polypeptides see below) or recombinant HIV-1 consensus clade B multimerizing polypeptides (MLB and MLD).
  • MVA polypeptides recombinant HIV-1 consensus clade B multimerizing polypeptides
  • MLB and MLD recombinant HIV-1 consensus clade B multimerizing polypeptides
  • Mouse sera were serially diluted in blocking buffer in duplicates and incubated overnight at 4°C. Plates were washed with PBS and bound antibodies targeting Env were determined by incubating with anti-mouse IgG secondary antibody conjugated with horseradish peroxidase diluted in blocking buffer for 3 hours. Plates were washed and the signal was developed with 0-phenylenediamine-H202 substrate. The reaction was stopped with 1M sulphuric acid. The absorbance was quantified at 492 nm by ELISA reader.
  • Plates were coated with the selected HIV-1 pseudotyped virus or recombinant HIV-1 multimerizing gpl20 protein as described in the previous method of Env-specific binding antibodies determination. 10E8, PGT126, or PGT121 antibodies were serially diluted in blocking buffer and applied in doublets with mouse hyperimmune sera diluted accordingly. Plates were washed and bound mouse antibodies were detected by rabbit anti-mouse IgG secondary antibody conjugated with horseradish peroxidase diluted in blocking buffer. Signal was developed and measured as described above.
  • Pseudotyped viruses were prepared using HEK293/17 cell line grown in 75 cm2 at 60-80% confluency co -transfected with 4 pg of plasmid coding Env and 8 pg of plasmid coding pSG3deltaEnv mixed with 48 pi of transfection reagent FuGene6 (Promega, Madison, WI, USA) in 12 ml of culture medium. After 2 days, produced pseudotyped viruses in culture medium were harvested, filtered and stored at 80°C until used.
  • Virus neutralization assay was done using various pseudotyped viruses of clades A, B, C, D and AE.
  • Murine leukemia virus (MULV)-pseudotyped virus containing the Env of MULV with the same backbone vector as all HIV- 1 -pseudotyped viruses (pSG3AEnv) was used as a negative control.
  • mouse sera were heat-inactivated for 1 hour at 56°C.
  • Sera in duplicates were serially diluted in 100 pi of culture medium in 96-well plates and incubated with 50 pi of pseudotyped viruses at 150.000 RLU for 90 minutes at 37°C.
  • the protein variants MLB036, MLB041, MLD033, MLD108 (His6-Myo-Flag) were produced in E.coli BL21 and in vivo biotinylated variants MLD068 and MLD049 (His6-Myo-Avi) in E.coli BL21 BirA strain. Proteins were purified using Ni-NTA agarose and assayed in ELISA. Increasing concentration of gpl20 inhibits binding of the MLB and MLD proteins at a constant concentration mM to bnAb.
  • mice were immunized by the administration of four doses of individual MLB variants including wild- type (MyoWT). Following immunization, sera were collected and tested in their reactivity with multimerizing HIV-1 gpl20 clade B consensus coated on ELISA plates. Antibody titers of IgG isotype were measured in ELISA. Statistical comparison was performed by ANOVA Kruskal- Wallis test with Dunn's post-test (* P ⁇ 0.05). Results are shown in Fig. 10. Binding of sera from mice immunized with MLB variants to HIV-1 multimeric gpl20 is specifically inhibited by PGT126.

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Abstract

La présente invention concerne des polypeptides ayant une longueur allant jusqu'à 180 acides aminés et contenant une séquence choisie parmi des séquences identiques ou différant au plus par 5 acides aminés par rapport à la séquence : KSELAVEILEKGQVRFWMQAX21X22X23X24GNAKVNYIFNEKEIFEGPKYKMHIDXsoXsiXszGIIEMFMEKLQDEDEGTYTFQLQX76X77X78X79X80NHSTVVLVGDVFKKLQKEAEFQRQEWIRKQG (SEQ ID No. 2), dans laquelle X21X22X23X24 est choisie parmi KAQQ (SEQ ID No. 33), LSVF (SEQ ID No. 34), ATPS (SEQ ID No. 35), EIMW (SEQ ID No. 36), DGSS (SEQ ID No. 37), LLPL (SEQ ID No. 38), WMWW (SEQ ID No. 39), MNLY (SEQ ID No. 40), MWRN (SEQ ID No. 41), IMME (SEQ ID No. 42), KHQL (SEQ ID No. 43), HWQF (SEQ ID No. 44), YAGN (SEQ ID No. 45) et HGQW (SEQ ID No. 46) ; X50X51X52 est choisie parmi RNT, IMF, GHE, PSW, RAN, YFW, ITL, QAM, DMR, WLW, QGE, VQY et VSL ; X76X77X78X79X8O est choisie parmi SHHLG (SEQ ID No. 47), FMLMM (SEQ ID No. 48), VILIL (SEQ ID No. 49), IVTPL (SEQ ID No. 50), DFIIW (SEQ ID No. 51), MWSE (délétion) (SEQ ID No. 52), LYYAW (SEQ ID No. 53), MMIEY (SEQ ID No. 54), WMTQT (SEQ ID No. 55), PQLWL (SEQ ID No. 56), EPIFL (SEQ ID No. 57) et QTATY (SEQ ID No. 58) ; et comprenant éventuellement en outre une étiquette d'affinité fixée à l'extrémité N-terminale ou à l'extrémité C-terminale de la séquence. Ces polypeptides imitent les épitopes de glycoprotéines Env du VIH-1 et sont utiles dans la préparation d'anticorps et de vaccins.
PCT/CZ2022/050017 2021-02-26 2022-02-18 Polypeptides imitant les épitopes de glycoprotéines env du vih-1 à boucle v3 et mper WO2022179648A1 (fr)

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CN202280017607.3A CN116964080A (zh) 2021-02-26 2022-02-18 模拟MPER和V3-环HIV-1 Env糖蛋白表位的多肽
US18/278,208 US20240226269A9 (en) 2021-02-26 2022-02-18 Polypeptides mimicking mper and v3-loop hiv-1 env glycoprotein epitopes
BR112023016617A BR112023016617A2 (pt) 2021-02-26 2022-02-18 Polipeptídeo, conjugado do polipeptídeo, vacina para a prevenção de infecção pelo vírus da imunodeficiência humana 1, vacina para o uso na prevenção de infecção pelo vírus da imunodeficiência humana 1, sequência de ácido desoxirribonucleico complementar, e, célula hospedeira
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WO2024091976A1 (fr) * 2022-10-24 2024-05-02 Duke University Compositions comprenant des peptides de région externe proximale de membrane de vih-1 (mper) et des acides nucléiques codant pour des peptides de mper
WO2024091968A1 (fr) * 2022-10-24 2024-05-02 Duke University Compositions comprenant des arnm codant pour des peptides de la région externe proximale de la membrane du vih-1 (mper)

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